Cell Signaling

Bruno Sopko

• Signal Transduction Pathways• Organization• Signals• Receptors– Soluble Receptors– Transmembrane Receptors

• Enzyme Coupled Receptors• G-Protein Coupled Receptors• Ion-Channel Coupled Receptors

• Second Messengers, Amplifiers, Integrators• Response Changes to Signals• Inhibitors

Content

Signal Transduction Pathways

Signal Transduction Pathways

Signals

Receptors

• Soluble Receptors• Transmembrane Receptors– Enzyme Coupled Receptors– G-Protein Coupled Receptors– Ion-Channel Coupled Receptors

Soluble Receptors - The steroid/Thyroid Hormone Superfamily of receptors

Cortisol (glucocortikoid) Androsteron (steroid)

All-trans retinol acid (retinoid)Vitamin D2

3,3',5-trijodo-L-thyronine (thyroid)

Soluble Receptors - The steroid/Thyroid Hormone Superfamily of receptors

Soluble Receptors - The steroid/Thyroid Hormone Superfamily of receptors

Transmembrane Receptors - Enzyme Coupled Receptors

• Tyrosine kinases phosphorylate protein tyrosine residues using ATP.

• Phospholipase C cleaves PIP2 into IP3 and DAG.

Tyrosine kinases / RAS MAP kinases

Tyrosine kinases / RAS MAP kinases

Tyrosine kinases / RAS MAP kinases

Tyrosine kinases / RAS MAP kinases

Tyrosine kinases / RAS MAP kinases

Tyrosine kinases / RAS MAP kinases

Tyrosine kinases / RAS MAP kinases

Tyrosine kinases / RAS MAP kinases

Tyrosine kinases / RAS MAP kinases

Tyrosine kinases / Insuline receptor

Tyrosine kinases / JAK-STAT receptors

JAK – Janus KinaseSTAT – Signal Transducer and Activator of Transcription

Tyrosin kinases / Receptors of Serin-Threonin Kinases

Cytokins mostly

Transmembrane Receptors / G-Protein Coupled Receptors

Charakteristic receptor structure

Heptahelical receptors (7 transmembrane α-helixes)

Transmembrane Receptors / G-Protein Coupled Receptors

Transmembrane Receptors / Ion Channel Coupled Receptors

Second messengers

• cAMP• cGMP• Phospholipids and Ca2+

• PI3 kinase/AKT and mTOR• MAP kinase pathway

cAMP

cAMP

cAMP

cGMP

Phosholipids and Ca2+

Phospholipids and Ca2+

Phospholipase C

Phosholipids and Ca2+

PI3 kinase/AKT and mTOR

MAP kinase pathway

Signalling molecules – vesicle stored and releasedSNAP-SNARE proteins

Signalling molecules – directly synthesized (Eicosanoids ...)

• compounds containing a 20-carbon core

• Members of this group:– prostaglandins– prostacyclines– tromboxanes– leukotrienes– lipoxins– hydroxyeicosatetraenoic acids (HETE)– hepoxilins

Eicosanoids biosynthesis

• A path in metabolism of polyunsaturated fatty acids (PUFAs), mainly linoleic and arachidonic acid arachidonic acid is (in humans) synthesized from linoleic acid:

• !!! it is not possible to synthesise de novo

• Most animals cannot form double bonds behind position ∆9linoleic and linolenic acids are essential: must be taken from food (plant oils, peanuts, soya beans, maize)

Eicosanoids biosynthesis - overview

Main eicosanoid production sites

• Endothelial cells • Leukocytes• Platelets• Kidneys

• Unlike e.g. histamin, eicosanoids are not synthesized in advance and stored in granules

• In case of an emergent need, these are rapidly produced from a released arachidonate

• Eicosanoids biosynthesis takes place in every cell type except red blood cells

Main steps of eicosanoids production

1) Activation of phospholipase A2 (PLA2)

2) Release of arachidonate into cytosol from membrane phospholipids by PLA2

3) Eicosanoids synthesis from arachidonate COX or LO pathway + further modifications by synthases/isomerases (PGH2

conversion to other prostanoids, LTA4 conversion..) depending on cell type

• PLA2 expression / activity stimulate:– interleukin-1– angiotensin II– bradykinin– thrombin– epinephrine…

1) Activation of phospholipase A2• Ca2+ dependent

• PLA2 expression / activity block:– dexamethasone (synthetic

corticoid)– annexin 1 (lipocortin) –

protein inducible by glucocorticoids

– caspase-3

dexamethasone

2) Arachidonate mobilization for eicosanoid synthesis

• From membrane phospholipids mostly by the action of phospholipase A2:

Release of arachidonate from phospholipids is blockedby anti-inflammatory steroids!

Eicosanoids biosynthesis

• 3 pathways:– A) cyclooxygenase – produces prostaglandins and

thromboxanes– B) lipoxygenase – produces leukotrienes, lipoxins,

hepoxilins and 12- and 15-HETE (hydroxyeicosatetraenoic acids)

– C) cytochrome P450 enzymes (monooxygenases) – produces HETE, e.g. 20-HETE; it is a main pathway in kidney proximal tubules

Products of COX pathway

• (thromboxane)

• (thromboxane)

• (prostacyclin)

Inhibition of COX pathway

Aspirin inhibits cyclooxygenase activity of PGHS-1 i PGHS-2 (by acetylation of enzyme serine)

Other non-steroidal anti-inflammatory drugs inhibit cyclooxygenase activity (ibuprofen – competes with arachidonate)

Anti-inflammatory corticosteroids block PGHS-2 transcription

Corticosteroids

Lipooxygenase pathway

• 3 different lipoxygenases indroduce oxygen to position 5, 12 or 15 in arachidonate; a primary product is hydroperoxy-eicosatetraenoic acid (HPETE)

• Only 5-lipoxygenase produces leuko-trienes; it requires protein FLAP

15-lipoxygenase

-GluLeukotriene D4 Leukotriene E4-Gly

peptidoleukotrienesGly–Cys–Glu

Hepoxilins(HXA3)

15-lipoxygenase12-lipoxygenase

5-lipoxygenase

15-lipoxygenase

5-lipoxygenase

Synthesis of eicosanoids by enzymes CYP450

• cytochrome P450 enzymes – monooxygenases: RH + O2 + NADPH + H+ ROH + H2O + NADP+

• Two types of compounds are produced:– epoxygenases - catalyse production of epoxyeicosatrienoic acids

(EETs) which are metabolized by epoxid-hydrolases into almost inactive dihydroxyeicosatrienoic acids (DiHETEs)

– hydroxylases - catalyse production of HETEs (20-HETE, 13-HETE etc.)

Eicosanoids - list

• arachidonic acid • CYP450

• DiHETEs

• 19-, 20-, 8-, • 9-, 10-, 11-, • 12-, 13-, 15-, • 16-, 17-,• 18-HETE

• cyklooxygenases

• prostacyklins

• prostaglandins

• tromboxanes

• lipoxygenases

• 5-, 8-, 12-, • 15-HETE

• lipoxins

• hepoxilins

• leukotrienes

EETs (epoxides)

Cytokines

• Group of proteins and peptides (glycopeptides) • Influence cell growth (growth factors) • Signal transmission from a cell to another cell

• Important group - lymphokines (also interleukins), proteins released from activated cells of immune system which coordinate immune response of the organism

Cytokine nomenclature• Lymphokines - produced by activated T-

lymphocytes, they control the response of immune system by signalization between immunocompetent cells

• Interleukins (IL) - target cells for IL are leukocytes • Chemokines - specific class, mediating chemotaxis

between cells; stimulate leukocyte movement and regulate their migration from blood into tissues

• Monokines - produced mainly by mononuclear cells, such as macrophages

Main function of cytokines

• Hematopoiesis (e.g. CSF - colony stimulating factor)

• Inflammatory reactions (e.g. IL1 - interleukin, TNF - tumor necrosis factor)

• Chemotaxis (e.g. IL8, MIP1- macrophage inflammatory protein 1, BLC – B-lymphocyte chemoatractant)

• Imunostimulation (e.g. IL12, IFNg - interferon)

• Imunosupression (e.g. IL10)

• Angiogenesis (e.g. VEGF- vascular endothelial growth factor)

• Embryogenesis (e.g. TGF-b, LT – lymphotoxin)

Signal termination

• The chemical messenger itself (acetylcholine esterase, insulin degradation in liver)

• The reaction itself (when GTP in G-protein is used, G-protein GDP complex forms the original structure)

• Degradation of second messenger (phosphodiesterase cleavage of cAMP)

• Phosphatases

Response Changes to Signals

• Intracellular Phosphorylation sites• Receptor number – downregulation• Hormone-receptor complex taken into cell by

endocytis• Degradation and recyclation of receptors• Number of available receptors can be altered

by other hormones

Response Changes to Signals

Literature

• R.K. Murray et al.: Harper's Illustrated Biochemistry, twenty-sixth edition, McGraw-Hill Companies, 2003

• Allan D. Marks, MD: Basic Medical Biochemistry a Clinical Approach, Lippincott Williams & Wilkins, 2009

• Ernst J. M. Helmreich, The Biochemistry of Cell Signalling, Oxford University Press, USA, 2001

• Geoffrey M. Cooper, Robert E. Hausman, The Cell: A Molecular Approach, Fourth Edition, Sinauer Associates, Inc., 2006

• Michael J. Berridge, Peter Lipp and Martin D. Bootman, The versatility and universality of calcium signalling, Nature Reviews | Molecular Cell Biology (1), 2000